Methods of signal generation and signal localization for improvement of signal readability in solid phase based bioassays

Abstract

A method for generating and localizing a signal in a solid phase substrate detection system comprises applying a solution of target material to a substrate; binding the target with a specific affinity molecule having an attached label, the label comprising multiple signal precursor molecules; applying a carrier to the substrate, and treating the label to convert the signal precursor molecules to signal generating molecules. The carrier comprises solvent for the label and thickener for localizing the signal. The carrier may include developer that converts signal precursor molecules to signal generating molecules. Developer is not necessary if the signal precursor molecules are converted to signal generating molecules by e.g. temperature change, pH change, sonication, light irradiation, microwave heating. A test device for detecting target in a fluid sample, and a kit of parts for determining the presence of target in a fluid sample are also disclosed.

The invention claimed is: 
     
       1. A lateral flow test device for detecting a target material in a fluid sample, said device comprising:
 a solid-phase substrate detection system comprising a porous membrane or a non-porous flat surface for transporting the fluid sample from a sample application site to a detection site; 
 disposed at a position remote from said detection site, a non-catalytic label conjugated with a specific affinity molecule for the target material, said non-catalytic label comprising a plurality of signal precursor molecules, said signal precursor molecules being convertible to a plurality of detectable signal generating molecules; 
 a carrier medium comprising a solvent for the dissolution of the non-catalytic label and a thickener for causing localization of signal generated by said plurality of detectable signal generating molecules at said detection site indicating the presence and/or quantity of said target material, and 
 an optically transparent lid for placement over the test strip after completion of a reaction to detect target material; 
 wherein said carrier medium is a continuous layer of gel located on the underside of said optically transparent lid and wherein said optically transparent lid is adapted to bring said continuous layer of gel into contact with said signal precursor molecules at least at said detection site. 
 
     
     
       2. The test device as claimed in  claim 1  wherein the optically transparent lid is hinged to a back laminate for holding porous components of the device. 
     
     
       3. The test device as claimed in  claim 1  further comprising a removable protective layer overlying the carrier medium. 
     
     
       4. The test device as claimed in  claim 1  wherein the porous membrane is selected from the group consisting of nitrocellulose and nylon. 
     
     
       5. The test device as claimed in  claim 1  wherein said affinity molecule is selected from the groups consisting of:
 (a) peptides or proteins selected from the group consisting of antibodies, genetically modified antibodies, monoclonal antibodies, polyclonal antibodies, receptors, antigens, lectins, avidins, oligopeptides, lipoproteins, glycoproteins, peptide hormones and allergens; 
 (b) nucleic acids selected from the group consisting of DNA, RNAs, oligonucleotides and aptamers; 
 (c) carbohydrates selected from the group consisting of mono-, oligo- and polysaccharides, glycolipids and proteo-polysaccharides; and 
 (d) low molecular weight ligands selected from the group consisting of biotin, biotin derivatives, steroids, hormones, cofactors, activators, inhibitors, drugs, allergens and haptens. 
 
     
     
       6. The test device as claimed in  claim 1  further comprising a signal developing reagent contained in said carrier medium for converting said plurality of signal precursor molecules to said plurality of detectable signal generating molecules. 
     
     
       7. The test device as claimed in  claim 6  wherein said signal developing reagent is a base or esterase. 
     
     
       8. The test device as claimed in  claim 1  wherein said signal precursor molecules are selected from the group consisting of fluorophores, luminophores, chromophores, prosthetic groups, redox active substances selected from the group consisting of redox mediators and electrode-active substances, bioluminogenic and fluorogenic proteins, visible dyes, fluorescent dyes, bioluminescent materials, chemiluminescent materials, electrochemically active materials, and magnetic materials. 
     
     
       9. The test device as claimed in  claim 8  wherein the signal precursor molecules are fluorophores selected from the group consisting of fluoresceins and their derivatives, fluorescein diacetate (FDA), fluorescein diacetate isothiocyanate (FDA-isothiocyanate), fluorescein diacetate maleimide (FDA-maleimide), cyanines, carbocyanines, rhodamines, xanthenes, diazo-dye based fluorescent substances, and small fluorescent aromatic and heteroaromatic molecules. 
     
     
       10. The test device as claimed in  claim 9  wherein said signal precursor molecules are selected from fluorescein diacetate (FDA), fluorescein diacetate isothiocyanate (FDA-isothiocyanate) and fluorescein diacetate maleimide (FDA-maleimide) and further comprising a signal developing reagent for the fluorescein diacetate (FDA), fluorescein diacetate Isothiocyanate (FDA-isothiocyanate) or fluorescein diacetate maleimide (FDA-maleimide). 
     
     
       11. The test device as claimed in  claim 8  wherein the signal precursor molecules are fluorescein diacetate (FDA), and wherein the carrier medium comprises a solvent for FDA, a signal developing reagent and a thickener. 
     
     
       12. The test device as claimed in  claim 11  wherein the solvent for FDA is dimethyl sulphoxide, the signal developing reagent is aqueous sodium hydroxide, and the thickener is Polysorbate 20. 
     
     
       13. The test device as claimed in  claim 8  wherein the signal precursor molecules are chromophores selected from the group consisting of cyanine, pyrazolone, anthraquinone, carbocyanine, rhodamine, xanthen, carotenoid and diazo- and monoazo, oxazine, indigoid, and riboflavine based dye substances. 
     
     
       14. The test device as claimed in  claim 1  wherein the proportion of thickener in the carrier medium ranges from 0.05 to 50% by weight based on the total weight of the carrier medium. 
     
     
       15. A test device of  claim 1  wherein the non-porous flat surface is selected from the group consisting of glass, polystyrene, metal and carbon.